Reversible protein Ser/Thr phosphorylation is a fundamental mechanism for cell regulation. While more than 400 Ser/Thr kinases have been identified in the human genome, there are only a few catalytic subunits for Ser/Thr phosphatases. In contrast to the previous assumption that phosphatases are constitutively active, recent work has shown that many phosphatases are highly regulated, largely through the formation of protein complexes with different regulatory or targeting subunits. This proposal focuses on the protein phosphatase 2A (PP2A), a central human phosphatase that regulates almost all aspects of cellular activity and is a critical tumor suppressor. Deregulation of PP2A is associated with breast, lung, and colorectal cancers as well as Alzheimer's Disease and susceptibility to viral and parasitic infection. A typical PP2A holoenzyme contains a scaffold A subunit, a catalytic C subunit and one of many regulatory B subunits, which are divided into B, B'and B" families. Despite the functional importance, it is still largely unknown how PP2A forms a functional complex and how the complex assembly is regulated. In this proposal, we aim to provide the structural basis for understanding the assembly and regulation of PP2A heterotrimeric complexes, through structural determination by X-ray crystallography and related biochemical analysis. Our study will be important not only for understanding the regulation of protein Ser/Thr dephosphorylation, but also for designing PP2A activators that either stabilize functional PP2A assembly or disrupt PP2A-inhibitory protein interactions. Such compounds can be useful for cancer treatment.